Mammalian DNA polymerase beta is an enzyme which catalyzes a central DNA replication and repair process in eucaryotic cells. As such a characterization of the structural mechanism of beta-Pol in regard to DNA-substrate binding is highly important for an understandIng of its function and/or dysfunction. Potential for misincorporations by beta-Pol within error-prone single-stranded DNA sequences or for template-primer substrates which contain DNA adducts relates directly to the mechanism of template primer DNA binding by this enzyme. These misincorporations usually result in mutations within genomic DNA. Multidimensional NMR structural studies are presently directed toward the N-terminal template binding domain of betaPol (residues 2-87). The abundantly expressed protein domain obtainable doubly labeled with 15N/13C is routinely overproduced and purified for these studies in a T7 overexpression system, which yields 40-50 mg of protein domain from 2 liters of culture medium. Double and triple resonance NMR experiments will be performed that allow assignment of 1H, 15N, and 13C chemical shifts within the protein domain. Structural data will be obtained by 15N and 13C-edited NOESY experiments in three and four dimensions. A highly refined NMR structure will be determined for the betaPol N-terminal domain both free in solution and in its complex with substrate DNA containing a 5mer of single-stranded nucleotides and either an upstream 3'-primer or a downstream 5'-phosphate "primer." The structure of the DNA complex (or the sites of interaction by nucleotides ff averaging is observed) will be similarly determined. A limited number of mutants defective in DNA binding will be structurally characterized after appropriate screening for loss of DNA binding is performed. The apparent free energy of residues contributing to template binding will be determined. From these results, an understanding of residues that contribute to template: binding, template alignment, and DNA polymerase fidelity will be achieved.